1,4–Diamino‐2‐butyne as the mechanism‐based pea diamine oxidase inhibitor

Abstract

1,4-Diamino-2-butyne is a mechanism-based inhibitor of diamine oxidase (EC 1.4.3.6) from pea cotyledons. It shows saturation kinetics K,,, = 1 mM like a substrate, but its interaction leads to time-dependent loss of enzyme activity which is not restored by gel filtration. The substrate 1,4-diaminobutane and the competitive inhibitor 1,4-diamin0-2-butanone protect the enzyme against inactivation. Changes in the enzyme electronic spectra with 1,4-diamino-2-butyne were found. The mechanism of the interaction involves an intermediate aminoallenic compound, which is formed with covalent bound pyrrole in the reaction of the nucleophile with the enzyme. The presence of a pyrrole in the inactivated enzyme was confirmed by reaction with Ehrlich's reagent. The kinetic data obtained in this study indicate that 1,4-diamino-2-butyne is a mechanism-based inactivator with number of turnovers, r = 17 and characteristic constants K' = 0.32 mM and k,, = 4.89 min-'.Mechanism-based inhibitors interact with enzymes by a mechanism involving a branched pathway, the branches representing turnover of substrate and inactivation of enzyme:Mechanism-based inactivator + enzyme e Complex I e Complex I1 + enzyme + product The action and kinetic behavior of these inactivators were described by Abeles [l] and Waley [2, 31. The mechanismbased inhibitors of beef plasma amine oxidase (Cu-containing) have been described. The oxidative mechanism of inactivation indicates that 2-bromoethylamine [l] is oxidized to bromoacetaldehyde bound at the active site. Many substituted allylamines [l, 41 act as mechanism-based inactivators of FADcontaining amine oxidases. These allylamines form reactive allenic compounds in the active site of the enzyme which react with the nucleophile in the active site and inactivate the enzyme. A similar mechanism has been suggested for beef plasma amine oxidase, which is inhibited by propargylamine, 1,4-diamin0-2-butyne and 2-chloroallylamine [5], but the exact mechanism has not ben confirmed. Also, (a-2-phenyl-3-chloroallylamine, which is a potent mechanism-based inactivator of FAD-containing monoamine oxidase, is a much wcaker inhibitor of hog kidney diamine oxidase (Cu-containing) [4]. Another type ofmechanism-based inactivators of beef plasma aniine oxidases are some esters with relatively acidic Enzymes. Diamine oxidase, diamine : O2 oxidoreductwe (deaminating, copper-containing) (EC 1.4.3.6); amino oxidase (flavincontaining) (EC 1.4.3.4); horseradish pcroxidase (EC 1.11.1.7); catalasc (EC 1.11.1.6).x-protons. These esters are converted to ketenes, which could acylate the active site and inactivate the enzyme [6]. Mechanism-based inactivators of plant Cu-containing amine oxidases have not been described previously.Pea diamine oxidase catalyzes the oxidation of many amines and diamines to aldehydes, H 2 0 2 and NH3; most readily oxidized are the diamines putrescine and cadaverine [7]. The ping-pong mechanism of this oxidation was described [8] and the presence of a cofactor with an o-quinone group in the catalytic cycle ...

1,5‐Diamino‐2‐pentyne is both a substrate and inactivator of plant copper amine oxidases

“…Similarly to the conversion of its lower homolog DABY by PSAO [7], DAPY oxidation by the studied enzymes led to their irreversible inhibition. The apparent inactivation constants K I of 10 )5 M are on the same order of magnitude as those K I values previously described for BEA [6] and DABY [7] in the reactions with lentil seedling amine oxidase (LSAO) and PSAO, respectively.…”

“…Lysine residues in plant CAOs are possible targets for covalent binding of reactive electrophilic aminoaldehydes formed by the turnover of acetylenic diamine substrates [7][8][9][10][11]. The cDNA of GPAO subunit (without the signal peptide) has been cloned and sequenced (D. Kopecˇny´, N. Houba-He´rin, H. G. Faulhammer & M. Sˇebela, unpublished results; EMBL/GenBank accession number AJ786401).…”

“…Kovacs' reagent containing DMAB (detects indoles and pyrroles [25]) was previously used for the visualization of pyrrole derivatives formed by the oxidation of DABY by plant amine oxidases [7,8]. Reaction mixtures of the studied CAOs with DAPY reacted positively with Kovacs' reagent after a period of incubation and provided a red soluble adduct upon heating.…”

“…It was then cooled and 2.5 mL of acetic acid was added to make up the volume to 6 mL. (c) Reaction with Kovacs' reagent: an aliquot (1 mL) of GPAO/ DAPY mixture (2 lM GPAO, 0.2 mM DAPY; initial concentrations) in 0.1 M potassium phosphate buffer, pH 7.0, was removed after 90 min of incubation at 23°C and mixed with 2 mL of the original Kovacs' reagent containing DMAB [7,8,25] (or its alternative contaning DMAC [8]), incubated at 50°C for 30 min and cooled on ice bath. In an alternative experiment, the GPAO/DAPY reaction mixture was first separated by ultrafiltration using the Microcon centrifugal cartridge as described above and only the ultrafiltrate mixed with Kovacs' reagent.…”

“…Among them, a special place is reserved for mechanismbased inhibitors, which undergo turnover-dependent conversion to electrophilic products capable of covalent binding to an active-site nucleophile resulting in inactivation. Two reported strategies in designing such inhibitors are the incorporation of either halogen or unsaturation at the b-position of amine substrates, examples being 2-bromoethylamine (BEA) [6] and 1,4-diamino-2-butyne (DABY) [7], respectively. Namely, the inactivating effect of DABY (as a putrescine analog) on plant CAOs has been studied in detail at the molecular level [8][9][10].…”